Viral and Nonviral Transfer of Genetic Materials to Adipose Tissues: Toward a Gold Standard Approach

Abstract

Gene transfer using viral or nonviral vectors enables the ability to manipulate specific cells and tissues for gene silencing, protein overexpression, or genome modification. Despite the widespread application of viral-and non-viral-mediated gene transfer to liver, heart, skeletal muscle, and the central nervous system, its use in adipose tissue has been limited. This is largely because adipose tissue is distributed throughout the body in distinct depots and adipocytes make up a minority of the cells within the tissue, making transduction difficult. Currently, there is no consensus methodology for efficient gene transfer to adipose tissue and many studies report conflicting information with regard to transduction efficiency and vector biodistribution. In this review, we summarize the challenges associated with gene transfer to adipose tissue and report on innovations that improve efficacy. We describe how vector and route of administration are the two key factors that influence transduction efficiency and outline a "gold standard" approach and experimental workflow for validating gene transfer to adipose tissue. Lastly, we speculate on how CRISPR/Cas9 can be integrated to improve adipose tissue research.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

“…After validating sgRNA efficiencies in vitro, AAV expression vectors were used to generate AAVs encoding a sgRNA and mCherry fluorescent marker. For transducing adipose tissues, AAV serotype 8 (AAV8) has been used most extensively owing to its favorable tropism for brown and white adipocytes (27)(28)(29)32,(41)(42)(43)(44)(45)(46).…”

“…Although AAV8 is the gold standard for transducing adipose tissues, its relatively small genome size (~4.7 kb) limits the ability to package S. pyogenes Cas9 with the sgRNA and mCherry transgene (27)(28)(29). Therefore, we developed a mouse line that expresses Cas9 specifically in brown adipocytes (BAd-CRISPR) by breeding two existing mouse lines: Ucp1 promoter-Cre recombinase (B6.FVB-Tg(Ucp1-cre)1Evdr/J) and Credependent Cas9-GFP (Rosa26-LSL-Cas9) (Supplemental Figure 2a) (10,24).…”

“…Initially, we attempted tail vein injection or intraperitoneal injection of 100 μL 10 12 vg/mL AAV8-mCherry to transduce all adipose tissues; however, both methods were unsuccessful and led to substantial transduction of liver 1 . However, direct injection of AAV8 into adipose depots has been shown to significantly improve transduction efficiency and reduce vector accumulation in liver, heart, and lung (27)(28)(29)41). To test this approach, we directly injected 100 μL 10 12 vg/mL AAV8-mCherry into interscapular BAT or psWAT and sacrificed mice after 14 days in accordance with previously established protocols (41,47).…”

“…Although AAV-mediated CRISPR/Cas9 gene transfer has proven useful for modeling disease in numerous tissues, its use in adipose tissues has been limited. This, in part, is because adipose tissues are located throughout the body, making transduction difficult (27)(28)(29). Moreover, mature adipocytes only comprise 11-40% of the total cell population of adipose tissues and lack unique cell surface receptors, which makes it challenging for vectors to navigate specifically to the adipocytes (27)(28)(29).…”

“…This, in part, is because adipose tissues are located throughout the body, making transduction difficult (27)(28)(29). Moreover, mature adipocytes only comprise 11-40% of the total cell population of adipose tissues and lack unique cell surface receptors, which makes it challenging for vectors to navigate specifically to the adipocytes (27)(28)(29). Currently, mutagenesis by CRISPR/Cas9 has been accomplished in preadipocyte culture models and in posterior subcutaneous WAT (psWAT).…”